The balance between macrophage cholesterol loading and efflux is an important determinant in the development of early atherosclerotic foam cell lesions in the arterial intima. The goals of this project are to identify the major pathways of cholesterol loading of macrophages in apoE deficient mice, to characterize the macrophage apolipoprotein receptor involved in cholesterol efflux from macrophages, and to probe the interactions amongst key molecules involved in HDL-mediated cholesterol efflux from macrophages. The role of these lipoprotein uptake and efflux pathways on the hypothesis that macrophages take up apoE- deficient betaVLDL via the recently cloned apoB48 receptor (B48r), not related to known proteins, which recognizes the amino terminal portion of apoB. We have created a mouse with this gene knocked out, and will create a transgenic mouse that expressed the B48r in the liver. We will breed these mice onto the apoE-deficient background and test the ability of the knockout macrophages to take up apoE-deficient betaVLDL, and the effect of these genetic manipulations on lipoprotein metabolism and atherosclerosis. In the Aim 2, we will test the hypothesis that macrophage efflux to lipid-free apolipoproteins occurs via a pathway utilizing the recently described Tangier disease gene, ABC-1. Utilizing the RAW macrophage cell line in which lipid efflux to, and the binding, uptake, and resecretion of apoAI or apoE is inducible by cAMP analogues, we will determine if ABC1 itself serves as an apolipoprotein receptor, or if it works in conjunction with another protein which serves this function. If the latter if found, we propose to identify the gene that encodes this apolipoprotein receptor. We will make macrophage specific knockouts and transgenics to probe the role of ABC1 and/or the putative apolipoprotein receptor on cholesterol efflux from primary macrophage cultures, and atherosclerosis in vivo. In Aim 3, we will test the hypothesis that there may be interactions between the pathways of macrophage lipid efflux to HDL or apoAI mediated by ABC-1, SRB1, apoE, and NPC. Transfections will test for interactions of apoE with SRB1, and SRB1 with ABC1. Macrophages from NPC mice will be tested for lipid efflux to HDL and apoAI. Confocal microscopy will be used to test for co- localization of these proteins during lipid efflux and determine if this process involves endocytosis and resecretion of HDL or apoAI.